Effects of Estrogen, Glucocorticoid, Glucagon, and Adenosine 3’ :5’-Monophosphate on Catalytic Activity, Amount, and Rate of de Nouo Synthesis of Hepatic Histidase*

The mechanisms by which estrogen, glucocorticoid, glucagon, and adenosine 3’:5’-monophosphate (CAMP), regulators which participate in the postnatal development of rat liver histidase, elevate the catalytic activity of this enzyme have been explored. A monospecific antibody against homo-geneously purified preparations of rat liver histidase has been elaborated in the goat. Employing this antibody in immunotitration experiments, it has been demonstrated that the elevations of hepatic histidase activity elicited by administration in uiuo of estradiol-17/3, cortisol acetate, glucagon, and N”,O”-dibutyryl adenosine 3’:5’-monophos-phate (dibutyryl CAMP) are paralleled, in each instance, by equivalent increments in immunoprecipitable histidase protein. Following administration of each of the three hormones and dibutyryl CAMP, rates of [Wlleucine incorporation

, and amino acids (4,5,8,9)) and others of which suppress histidase activity (e.g. pituitary components (10, 111, androgen (121, and thyroxine (13, 14) from the linear portions of the curves, in the region of zero order kinetics, using an extinction coefficient of 18,800 for the reaction product, urocanate (22). One unit of enzyme activity is defined as the amount producing 1 pmol of urocanic acid/min under the conditions described.
The sensitivity of this assay was 0.005 unit of activity.
Specific activity is defined as enzyme units per mg soluble protein, as measured by the method of Lowry et al. (23).
Acrylamide Gel Electrophoresis -Analytical acrylamide gel electrophoresis was carried out according to the procedure of Davis (24) in 7.5% gels at pH 8.3 at 4". A current of 3 mA/gel was applied for 3 h. Gels were stained for protein in 0.5% aniline blue black in 7% acetic acid for 1 h.
Histidase activity was detected on gels immediately following electrophoresis by soaking the unstained gel in 0.1% eosin followed by immersion in the histidase assay mixture for approximately 20 min (25). Eosin fluoresces when excited with a 254 nm ultraviolet light source; the nonfluorescent (quenched) band on the gel corresponds to urocanate.
Preparation of a Monospecific Antibody Prepared against Rat Liver Histidase-Antiserum to rat liver histidase was obtained in the following manner. Purified homogenous histidase (250 to 300 pg) was applied to an acrylamide gel (1 x 10 cm) and electrophoresed at 15 mA/tube for 3 h, as described above. A thin longitudinal gel slice was removed and stained for enzymatic activity, as described above. The unstained gel segment corresponding to the histidase activity was sliced transversely, homogenized in 3 to 4 ml of 0.15 M NaCl, and emulsified in an equal volume of complete Freund's adjuvant. This suspension was injected subcutaneously into a goat and was followed by a second injection 6 weeks after the first and a series of booster injections at 2-to g-month intervals. Highest antibody titers were A portion of each homogenate was centrifuged at 122,000 x g for 60 min and used for determination of incorporation of radioactivity into total protein.
One-half milliliter of these high speed supernatants was precipitated with 3 ml of 10% trichloroacetic acid, the precipitates were washed three times with 3 ml of 10% trichloroacetic acid and dissolved in 0.5 N NaOH for radioactive counting in Bray's solution (27)

Hormonal
Effects on Hepatic Histidase Synthesis

RESULTS
Characteristics of Purified Rat Liver Histidase and Its Antibody -Disc electrophoresis of freshly prepared native enzyme (Table I, Step 7) on nondenaturing acrylamide gels revealed a single protein band upon aniline blue black staining (Fig. 1). Measurement of histidase activity on such gels, by the eosin-staining technique, demonstrated one enzyme activity band which corresponded to the protein-stained band. Liver histidase, as purified by the above described procedures, thus migrates as a single homogeneous enzymatic species, free of protein contaminants.
That this enzyme preparation has been purified to homogeneity has been further affirmed by the findings of: constant high specific activities in the most active fractions of the final purification step, hydroxylapatite chromatography, and upon rechromatography on DEAE-cellulose; a single subunit band following sodium dodecyl sulfate-polyacrylamide electrophoresis; and sedimentation as a single species following analytical ultracentrifugation.2 Immunoglobulin preparations of goat antisera withdrawn following immunization with purified histidase preparations were tested for monospecificity in the Ouchterlony double diffusion system. Antibody preparations were obtained which yielded a single precipitin band, when diffusing against a high speed supernatant of female rat liver homogenate; this band fused with a reaction of identity with that of purified histidase (Fig. 2). These antibody preparations were thus monospecific. Moreover, unfractionated cytosols and purified histidase prepared from male rat liver also yielded single precipitin bands, which formed lines of identity with each other and with the female enzyme. Thus, although hepatic histidase activity in the adult female liver is more than double that in male (3), histidases present in the livers of both sexes are immunologically identical.
FIG. 1. Disc acrylamide gel electrophoresis of purified histidase from female rat liver. Thirty micrograms of purified native enzyme was electrophoresed on a 7.5% nondenaturing acrylamide gel.

Effects of Estrogen, Glucocorticoid,
Glucagon, and Cyclic AMP on Amounts of Histidase Protein - Table  II depicts a typical experiment in which administration in vivo of eight daily doses of glucagon, dibutyryl CAMP (plus theophylline), estradiol-17/3, or cortisol acetate, in triplicate animals, elevated hepatic histidase activity in B-day-old male rats to 167, 122, 200, and 152%, respectively, of control values. In an attempt to determine whether these hormonal elevations in enzyme activities are due to corresponding increases in amounts of enzyme protein, increasing amounts of heattreated liver cytosols from these animals were immunotitrated with constant amounts of anti-histidase, as described under "Materials and Methods." Fig. 3 illustrates that identical equivalence points were observed with liver preparations from untreated control animals and those receiving estradiol-17P, cortisol acetate, glucagon, or dibutyryl CAMP (plus theophylline), in whom histidase activity levels were elevated. In each case, a given quantity of antibody precipitated identical amounts of catalytic activity, following which further added histidase was recoverable in the supernatant.
Furthermore, measurement of the protein content of the immunoprecipitates indicated that, in each case, as the antigen/antibody ratio increased progressively, increasing amounts of protein were precipitated, followed by a plateau at the equivalence zone, then a decrease in precipitable protein in the region of antigen excess. The point of maximum protein precipitation was in good agreement with that of the equivalence point, as determined by supernatant enzyme assays; approximately equal amounts of protein were precipitated per unit of catalytic  (Table I; Step 7); C, 2.5 x 1O-3 units of analoaouslv purified histidase from male rat liver: andD, 1.5 x 10e3 units of a high speed supernatant of a male rat liver homogenate.
activity under each experimental condition. It may be inferred from these two immunochemical measurements of histidase, that all elevations in histidase catalytic activity following treatment with estradiol-17P, cortisol acetate, glucagon, and CAMP are a result of proportionate increases in the quantity of immunologically identical histidase protein.   Table II. Constant amounts (0.125 ml) of anti-histidase immunoglobulin preparation were incubated with increasing amounts of heat-treated liver cytosols, containing the histidase activities indicated on the abscissa, and assayed for residual enzyme activity recoverable in the supernatants and precipitable protein, as described under "Materials and Methods." Each symbol represents the mean of results from triplicate animals.
whether the observed increases in catalytic activity and amount of enzyme protein following estradiol-17p, cortisol, glucagon, and CAMP administration are due to increased histidase biosynthetic rates, the amounts of L-[Wleucine incorporation in Go, during a 45min period, into immunoprecipitable histidase, relative to that into the total soluble protein, were compared in control and treated animals. Table III demonstrates that a doubling enzyme activity upon glucagon administration was accompanied by a comparable rise in L-[Wlleucine incorporation into histidase, relative to that into total soluble protein. Similarly, the 40% rise in enzyme activity following dibutyryl CAMP (plus theophylline) administration was paralleled by a 61% increase in relative synthetic rate of histidase. Furthermore, rats receiving estradiol-17g, whose enzyme activities are 164% of control values manifested a doubling in the rate of relative histidase synthesis. An increase of 76% in catalytic activity following cortisol acetate administration was accompanied by a 52% higher rate of relative histidase synthesis. Thus, all hormonally induced elevations in histidase catalytic activity and in amounts of enzyme protein are accompanied by approximately parallel increases in in vivo biosynthetic rate of the enzyme.
To evaluate the identity and purity of the immunoprecipitates, W-labeled immunoprecipitates were analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis. Fig. 4 depicts densitometric tracings and radioactive measurements of a typical stained sodium dodecyl sulfate electrophoretic gel. The only three protein peaks detectable in the immunoprecipitates, in order of decreasing molecular weight, were protein bands corresponding to the subunits of purified rat liver histidase and to the heavy and light immunoglobulin chains. Radioactivity measurements determined on gel slices indicated that the histidase subunit peak contained all of the radioactivity, the two immunoglobulin bands and the protein-free portions of the gel manifesting no detectable radioactivity.
Thus, virtually all of the radioactivity present in the immunoprecipitates is incorporated into the histidase molecule.

DISCUSSION
That elevations in catalytic activity of rat liver histidase elicited by estradiol-17B, cortisol acetate, glucagon, and dibutyryl CAMP are each accompanied by equivalent increments in quantities of immunologically identical enzyme protein has been demonstrated by immunotitration experiments, in which identical equivalence points were observed and equal quanti-